The apical sodium-dependent bile acid transporter (ASBT) plays a key role in the enterohepatic recycling of bile salts, cholesterol homeostasis, and serves as a molecular target for hypercholesterolemic agents. Although the transporter sequence is known, there is controversy about its membrane topology and very little is known about ASBT structure-function and ligand binding domains. The proposed research will focus on the structural biology of ASBT. Using a novel approach that combines molecular and computational biology our long-term goal is to delineate the three-dimensional structure, ligand-binding domains, and cellular transport mechanism of ASBT. The following specific aims will be addressed: (1) define the membrane topology of ASBT using a series of topology scanning approaches; (2) Construct a comprehensive structural and predictive model of ASBT that can correlate structural point mutations to changes in ligand affinity and transport; (3) Define the functional regions of ASBT by site-directed mutagenesis; we have developed a computer-assisted site-directed mutagenesis approach to probe ASBT protein for amino acid residues implicated in ligand and sodium interactions; (4) Determine the ligand binding domains of ASBT by mass spectrometry; we will employ selective photoaffinity labels to determine ligand-binding peptide sequences. Information gained by these studies will significantly increase our understanding of the structural interactions that drive bile acid transport and further our structural knowledge of solute carrier proteins in general. Additionally, it may aid future development of specific therapeutic strategies against hypercholesterolemia and related cardiovascular diseases.